Antibodies targeting egfr and use thereof

Abstract

Disclosed are proteins with antibody heavy chain and light chain variable domains that can be paired to form an antigen-binding site targeting EGFR on a cell, pharmaceutical compositions comprising such proteins, and therapeutic methods using such proteins and pharmaceutical compositions, including for the treatment of cancer.

What is claimed is: 
     
         1 . An antigen-binding site that binds EGFR, comprising:
 (i) a heavy chain variable domain (VH) comprising complementarity-determining region 1 (CDR1), complementarity-determining region 2 (CDR2), and complementarity-determining region 3 (CDR3) sequences of SEQ ID NOs: 2, 23, and 4, respectively; and a light chain variable domain (VL) comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 6, 7, and 17, respectively; or   (ii) a VH comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 2, 12, and 4, respectively; and a VL comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 6, 7, and 8, respectively.   
     
     
         2 . The antigen-binding site of  claim 1 , wherein the VH comprises CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 2, 23, and 4, respectively; and the VL comprises CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 6, 7, and 17, respectively. 
     
     
         3 . The antigen-binding site of  claim 2 , where the VH comprises CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 2, 12, and 4, respectively; and the VL comprises CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 6, 7, and 17, respectively. 
     
     
         4 . The antigen-binding site of  claim 2 , wherein the VH comprises CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 2, 3, and 4, respectively; and the VL comprises CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 6, 7, and 17, respectively. 
     
     
         5 . The antigen-binding site of  claim 1 , wherein the VH comprises CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 2, 12, and 4, respectively; and the VL comprises CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 6, 7, and 8, respectively. 
     
     
         6 . An antigen-binding site comprising: a VH comprising an amino acid sequence at least 90% identical to SEQ ID NO:1 and a VL comprising an amino acid sequence at least 90% identical to SEQ ID NO:5, wherein the VH comprises an S62R substitution relative to SEQ ID NO:1 and/or the VL comprises a D92R substitution and/or an F87Y substitution relative to SEQ ID NO:5, numbered under the Chothia numbering scheme. 
     
     
         7 . The antigen-binding site of  claim 6 , wherein the VH comprises an S62R substitution relative to SEQ ID NO:1, numbered under the Chothia numbering scheme. 
     
     
         8 . The antigen-binding site of  claim 6 , wherein the VL comprises a D92R substitution relative to SEQ ID NO:5, numbered under the Chothia numbering scheme. 
     
     
         9 . The antigen-binding site of  claim 6 , wherein the VH comprises an S62R substitution relative to SEQ ID NO:1 and the VL comprises a D92R substitution relative to SEQ ID NO:5, numbered under the Chothia numbering scheme. 
     
     
         10 . The antigen-binding site of any one of  claims 1 - 9 , wherein the VL comprises an F87Y substitution relative to SEQ ID NO:5, numbered under the Chothia numbering scheme. 
     
     
         11 . The antigen-binding site of any one of  claims 1 - 3 , and  6 - 10 , wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO:11 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 16. 
     
     
         12 . The antigen-binding site of any one of  claims 1 - 3 , and  6 - 11 , wherein the VH comprises the amino acid sequence of SEQ ID NO:11 and the VL comprises the amino acid sequence of SEQ ID NO:16, or the VH comprises the amino acid sequence of SEQ ID NO:32 and the VL comprises the amino acid sequence of SEQ ID NO:33. 
     
     
         13 . The antigen-binding site of any one of  claims 1 ,  2 ,  4 ,  6 ,  8 , and  10 , wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO:1 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO:16. 
     
     
         14 . The antigen-binding site of any one of  claims 1 ,  2 ,  4 ,  6 ,  8 ,  10 , and  13 , wherein the VH comprises the amino acid sequence of SEQ ID NO:1 and the VL comprises the amino acid sequence of SEQ ID NO:16. 
     
     
         15 . The antigen-binding site of  claim 1 ,  5 - 7 , and  10 , wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO:11 and the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 13. 
     
     
         16 . The antigen-binding site of  claim 1 ,  5 - 7 ,  10 , and  15 , wherein the VH comprises the amino acid sequence of SEQ ID NO:11 and the VL comprises the amino acid sequence of SEQ ID NO: 13. 
     
     
         17 . The antigen-binding site of any one of  claims 1 - 16 , wherein the antigen-binding site is present as a single-chain fragment variable (scFv), and wherein the scFv comprises a sequence selected from SEQ ID NOs: 14, 18, 20, and 24. 
     
     
         18 . The antigen-binding site of any one of  claims 1 - 17 , wherein the antigen-binding site binds human EGFR with a dissociation constant (K D ) smaller than or equal to 5 nM, as measured by surface plasmon resonance (SPR). 
     
     
         19 . The antigen-binding site of any one of  claims 1 - 18 , wherein the antigen-binding site binds rhesus macaque EGFR with a dissociation constant (K D ) smaller than or equal to 6 nM, as measured by surface plasmon resonance (SPR). 
     
     
         20 . A protein comprising the antigen-binding site of any one of the  claims 1 - 19 . 
     
     
         21 . The protein of  claim 20 , further comprising an antibody heavy chain constant region. 
     
     
         22 . The protein of  claim 21 , wherein the antibody heavy chain constant region is a human IgG heavy chain constant region. 
     
     
         23 . The protein of  claim 22 , wherein the antibody heavy chain constant region is a human IgG1 heavy chain constant region. 
     
     
         24 . The protein of  claim 22  or  23 , wherein each polypeptide chain of the antibody heavy chain constant region comprises an amino acid sequence at least 90% identical to SEQ ID NO:26. 
     
     
         25 . The protein of any one of  claims 22 - 24 , wherein at least one polypeptide chain of the antibody heavy chain constant region comprises one or more mutations, relative to SEQ ID NO:26, at one or more positions selected from Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and K439, numbered according to the EU numbering system. 
     
     
         26 . The protein of any one of  claims 22 - 25 , wherein at least one polypeptide chain of the antibody heavy chain constant region comprises one or more mutations, relative to SEQ ID NO:26, selected from Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D, and K439E, numbered according to the EU numbering system. 
     
     
         27 . The protein of any one of  claims 22 - 26 , wherein one polypeptide chain of the antibody heavy chain constant region comprises one or more mutations, relative to SEQ ID NO:26, at one or more positions selected from Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, K392, T394, D399, 5400, D401, F405, Y407, K409, T411 and K439; and the other polypeptide chain of the antibody heavy chain constant region comprises one or more mutations, relative to SEQ ID NO:26, at one or more positions selected from Q347, Y349, L351, S354, E356, E357, S364, T366, L368, K370, N390, K392, T394, D399, D401, F405, Y407, K409, T411, and K439, numbered according to the EU numbering system. 
     
     
         28 . The protein of  claim 27 , wherein one polypeptide chain of the antibody heavy chain constant region comprises K360E and K409W substitutions relative to SEQ ID NO:26; and the other polypeptide chain of the antibody heavy chain constant region comprises Q347R, D399V and F405T substitutions relative to SEQ ID NO:26, numbered according to the EU numbering system. 
     
     
         29 . The protein of  claim 27  or  28 , wherein one polypeptide chain of the antibody heavy chain constant region comprises a Y349C substitution relative to SEQ ID NO:26; and the other polypeptide chain of the antibody heavy chain constant region comprises an S354C substitution relative to SEQ ID NO:26, numbered according to the EU numbering system. 
     
     
         30 . An antibody-drug conjugate comprising the protein of any one of  claims 20 - 29  and a drug moiety. 
     
     
         31 . The antibody-drug conjugate of  claim 30 , wherein the drug moiety is selected from the group consisting of auristatin, N-acetyl-7 calicheamicin, maytansinoid, pyrrolobenzodiazepine, and SN-38. 
     
     
         32 . An immunocytokine comprising the antigen-binding site of any one of  claims 1 - 19  and a cytokine. 
     
     
         33 . The immunocytokine of  claim 32 , wherein the cytokine is selected from the group consisting of IL-2, IL-4, IL-10, IL-12, IL-15, TNF, and IFNα. 
     
     
         34 . A bispecific T-cell engager comprising the antigen-binding site of any one of  claims 1 - 19  and an antigen-binding site that binds CD3. 
     
     
         35 . A chimeric antigen receptor (CAR) comprising:
 (a) the antigen-binding site of any one of  claims 1 - 19 ;   (b) a transmembrane domain; and   (c) an intracellular signaling domain.   
     
     
         36 . The CAR of  claim 35 , wherein the transmembrane domain is selected from the transmembrane regions of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, EGFR, CD37, CD64, CD80, CD86, CD134, CD137, CD152, and CD154. 
     
     
         37 . The CAR of  claim 35  or  36 , wherein the intracellular signaling domain comprises a primary signaling domain comprising a functional signaling domain of CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. 
     
     
         38 . The CAR of any one of  claims 35 - 37 , wherein the intracellular signaling domain further comprises a costimulatory signaling domain comprising a functional signaling domain of a costimulatory receptor. 
     
     
         39 . The CAR of  claim 38 , wherein the costimulatory receptor is selected from the group consisting of OX40, CD27, CD28, CD30, CD40, PD-1, CD2, CD7, CD258, NKG2C, B7-H3, a ligand that binds to CD83, ICAM-1, LFA-1 (CD11a/CD18), ICOS and 4-1BB (CD137), or any combination thereof. 
     
     
         40 . An isolated nucleic acid encoding the CAR of any one of  claims 35 - 39 . 
     
     
         41 . An expression vector comprising the isolated nucleic acid of  claim 40 . 
     
     
         42 . An immune effector cell comprising the nucleic acid of  claim 40  or the expression vector of  claim 41 . 
     
     
         43 . An immune effector cell expressing the CAR of any one of  claims 35 - 39 . 
     
     
         44 . The immune effector cell of  claim 42  or  43 , wherein the immune effector cell is a T cell. 
     
     
         45 . The immune effector cell of  claim 44 , wherein the T cell is a CD8 +  T cell, a CD4 +  T cell, a γδ T cell, or an NKT cell. 
     
     
         46 . The immune effector cell of  claim 42  or  43 , wherein the immune effector cell is an NK cell. 
     
     
         47 . A pharmaceutical composition comprising the protein of any one of  claims 20 - 29 , the antibody-drug conjugate of  claim 30  or  31 , the immunocytokine of  claim 32  or  33 , the bispecific T-cell engager of  claim 34 , or the immune effector cell of any one of  claims 42 - 46 ; and a pharmaceutically acceptable carrier. 
     
     
         48 . A method of treating cancer, the method comprising administering to a subject in need thereof an effective amount of the protein of any one of  claims 20 - 29 , the antibody-drug conjugate of  claim 30  or  31 , the immunocytokine of  claim 32  or  33 , the bispecific T-cell engager of  claim 34 , or the immune effector cell of any one of  claims 42 - 46 , or the pharmaceutical composition of  claim 47 . 
     
     
         49 . The method of  claim 48 , wherein the cancer is a solid tumor. 
     
     
         50 . The method of  claim 48  or  49 , wherein the cancer is lung cancer, breast cancer, kidney cancer, colorectal cancer, gastric cancer, brain cancer, glioma, bladder cancer, head and neck cancer, bladder cancer, pancreatic cancer, and liver cancer, cervical cancer, ovarian cancer or prostate cancer. 
     
     
         51 . The method of any one of  claims 48 - 50 , wherein the cancer expresses EGFR. 
     
     
         52 . The antigen-binding site of any one of  claims 1 - 19 , the protein of any one of  claims 20 - 29 , the antibody-drug conjugate of  claim 30  or  claim 31 , the immunocytokine of  claim 32  or  claim 33 , or the bispecific T cell engager of  claim 34 , wherein the antigen-binding site, protein, antibody-drug conjugate, immunocytokine, or bispecific T cell engager is a purified antigen-binding site, protein, antibody-drug conjugate, immunocytokine, or bispecific T cell engager. 
     
     
         53 . The antigen-binding site, protein, antibody-drug conjugate, immunocytokine, or bispecific T cell engager of  claim 52 , wherein the antigen-binding site, protein, antibody-drug conjugate, immunocytokine, or bispecific T cell engager is purified by a method selected from the group consisting of: centrifugation, depth filtration, cell lysis, homogenization, freeze-thawing, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography.